Telomerase is an enzyme that synthesizes telomeres on chromosome ends. Telomeres are DNA sequences found at the ends of eukaryotic chromosomes which maintain the fidelity of genetic information during replication. Under normal circumstances, telomeres become shorter and shorter with each cycle of cell division. A sufficiently short telomere is believed to signal the cells to stop dividing.
Telomerase belongs to a class of enzymes known as reverse transcriptases that use RNA as a template for creating DNA. Telomerase contains both RNA and protein components. The RNA portion of the enzyme binds to the DNA in the telomere while the protein component lures DNA subunits into the region and attaches them to the end of the chromosome. Telomerase then elongates the G-rich strand of chromosomal termini by adding telomeric repeats. This elongation occurs by reverse transcription of a part of the telomerase RNA component, which contains a sequence complementary to the telomere repeat. Following telomerase-catalyzed extension of the G-rich strand, the complementary DNA strand of the telomere is presumably replicated by more conventional means. In the case of eukaryotic organisms, telomerases are composed of an accumulation of repeated defined nucleotide sequences (repeats) which, for example, contain the sequence TTAGGG in humans.
Telomerase activity is not detectable in normal tissues except germline cells. Germline cells, whose chromosomal ends must be maintained through repeated rounds of DNA replication, do not decrease their telomere length with time, presumably due to the activity of telomerase. Stem cells of renewing tissues express very low levels of telomerase and their telomeres shorten with multiple cell divisions. Telomerase activity is occasionally detected in tissues adjacent to tumors possibly reflecting the presence of occult micrometastases.
Telomerase is believed to have a role in the process of cell senescence. The repression of telomerase activity in somatic cells is likely to be important in controlling the number of times they divide. Indeed, the length of telomeres in primary fibroblasts correlates well with the number of divisions these cells can undergo before they senescence. The loss of telomeric DNA may signal to the cell the end of its replicative potential, as part of an overall mechanism by which multicellular organisms limit the proliferation of their cells.
Due to its role in controlling replication, telomerase has also recently been implicated in oncogenesis. Telomerase activity has been detected in most tumor cells. It has been suggested that telomerase is responsible for the unchecked growth of human cancer cells. Unlike normal cells, in cancer cells telomerase appears to grant the cell immortality by maintaining telomere length so that the cell never receives a signal to stop dividing. The telomerase enzyme is an ideal target for chemotherapy because this enzyme is active in about 90 percent of human tumors, but inactive in most normal cells. Pharmaceutical companies have screened thousands of compounds to find agents capable of blocking telomerase.
A method termed as telomeric repeat amplification protocol (TRAP) has been developed to measure telomerase activity. TRAP is based on the in vitro detection of the enzyme activity. Briefly, a synthetic oligonucleotide derived from the telomere sequence is used as a substrate. This substrate is elongated by the telomerase in a test sample and the elongation product is then amplified and quantified. Detailed description of the TRAP methods can be found in, for example, U.S. Pat. No. 5,891,639 to Harley et al. (hereinafter Harley) and U.S. Pat. No. 6,221,584 to Emrich et al. (hereinafter Emrich). Recently, a number of research groups have reported modified TRAP methods using real-time polymerase chain reaction (PCR) technology (See e.g., Hou et al., Clin. Chem. 47:519-524, 2001; Elmore et al., Diagn. Mol. Pathol., 11,177-185, 2002; and Wege et al., Nucleic Acids Res., 31:E3-3, 2003). Specifically, real-time PCR technology has been employed to provide a faster and more sensitive quantification of the elongation product of telomerase.
The current TRAPs typically include multiple incubation steps and transfer of sample from one tube to another after each incubation step. The transferring process is time consuming and prone to contamination and operation error (e.g., adding samples to a wrong tube or well). The current TRAPs usually start with a cell or tissue extract. Since telomerases, which contain both RNA and protein components, are subjected to the digestion of proteases and RNases in the extract, protease inhibitors and/or RNase inhibitors are often needed to prevent the degradation of the telomerases. Addition of protease inhibitors and/or RNase inhibitors to the extract increase the cost of the analysis. Thus, a need exists for an telomerase assay that is more flexible and can be performed easily and quickly at a low cost.